Peptides

ABSTRACT

The present invention relates to dual-site BACE1 inhibitors, their manufacture, pharmaceutical compositions containing them and their use as therapeutically active substances. The active compounds of the present invention are useful in the therapeutic and/or prophylactic treatment of e.g. Alzheimer&#39;s disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application of PCT/EP2015/077582filed Nov. 25, 2015, which claims priority from European PatentApplication No, 14195325.7, filed on Nov. 28, 2014. The priority of bothsaid PCT and European Patent Application are claimed. Each of priormentioned applications is hereby incorporated by reference herein in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Aug. 13, 2018, is named036844-94951_SL.txt and is 26,082 bytes in size.

The present invention is concerned with peptides having dual BACE1inhibitory properties, their manufacture, pharmaceutical compositionscontaining them and their use as therapeutically active substances.

The present invention is concerned with peptides having dual BACE1inhibitory properties, their manufacture, pharmaceutical compostiionscontaining them and their use as therapeutically active substances.

TECHNICAL FIELD

The present compounds have Asp2 (β-secretase, BACE1 or Memapsin-2)inhibitory activity and may therefore be used in the therapeutic and/orprophylactic treatment of diseases and disorders characterized byelevated β-amyloid levels and/or β-amyloid oligomers and/or β-amyloidplaques and further deposits, particularly Alzheimer's disease.

BACKGROUND ART

Alzheimer's disease (AD) is a neurodegenerative disorder of the centralnervous system and the leading cause of a progressive dementia in theelderly population. Its clinical symptoms are impairment of memory,cognition, temporal and local orientation, judgment and reasoning butalso severe emotional disturbances. There are currently no treatmentsavailable which can prevent the disease or its progression or stablyreverse its clinical symptoms. AD has become a major health problem inall societies with high life expectancies and also a significanteconomic burden for their health systems.

The BACE1 enzyme is responsible for one of the proteolytic cleavages ofthe APP protein that contributes to the generation of the Alzheimer'sdisease-associated Aβ-peptide. Retarding or stopping the production ofAO-peptide through inhibition of the BACE1 enzyme is a promisingtherapeutic concept.

Active site-directed BACE1 inhibitors are described in e.g.WO2006/002907 and exosite-directed (catalytic domain) BACE1 inhibitorsare described in e.g. Kornacker et al., Biochemistry 2005, 44, 11567-73.

Bodor et al describe modified peptides suitable to penetrate theblood-brain-barrier (Bodor et al., Science, Vol. 257, 1992).

DETAILED DESCRIPTION OF THE INVENTION

Object of the present invention is dual-site BACE1 inhibitor, binding toboth, the enzymatic active site and the catalytic domain of the BACEenzyme, the preparation of the above mentioned compounds, medicamentscontaining them and their manufacture as well as the use of the abovementioned compounds in the therapeutic and/or prophylactic treatment ofdiseases and disorders which are associated with inhibition of BACE1activity, such as Alzheimer's disease. Furthermore, the formation, orformation and deposition, of β-amyloid plaques in, on or aroundneurological tissue (e.g., the brain) are inhibited by the presentcompounds by inhibiting the Aβ production from APP or an APP fragment.

The following definitions of the general terms used in the presentdescription apply irrespectively of whether the terms in question appearalone or in combination with other groups.

TABLE 1 amino acid abbreviations used herein Amino Acid 3-Letter1-Letter Alanine Ala A D-Alanine DAla a Arginine Arg R Asparagine Asn NAspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln QGlycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine LysK D-Lysine DLys k Methionine Met M Norleucine Nle — Ornithine Orn OPhenylalanine Phe F Proline Pro P D-Proline DPro p Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

The term “Sta” stands for statine,(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid (CAS 49642-07-1).

The term “MetSta” stands for(3S,4S)-4-amino-3-hydroxy-6-methylthiohexanoic acid (CAS n/a), (CAS ofFmoc protected: 268542-18-3).

The term “27-OH-Chol” stands for 27-hydroxycholesterol (CAS 20380-11-4).Structure and preparation see page 18, compound 4.

The term “Chol-27-TFA-ester” stands for succinamic acid(3S,8S,9S,10R,13R,14S,17R)-17-[(1R,5R)-1,5-dimethyl-6-(2,2,2-trifluoro-acetoxy)-hexyl]-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester, this occurred as byproduct during TFA cleavage of“27-OH-Chol”-peptides.

The term “Chol‘ester”’ stands for cholesteryl hemisuccinate (CAS:1510-21-0).

The term “PEG(3) stands for 12-amino-4,7,10-trioxadodecanoic acid (CAS:784105-33-5).

The term “PEG(4) stands for 15-amino-4,7,10,13,tetraoxapentadecanoicacid (CAS: n/a), (CAS of Fmoc protected: 557756-85-1).

The term “pharmaceutically acceptable salts” refers to salts that aresuitable for use in contact with the tissues of humans and animals.Examples of suitable salts with inorganic and organic acids are, but arenot limited to acetic acid, citric acid, formic acid, fumaric acid,hydrochloric acid, lactic acid, maleic acid, malic acid,methane-sulfonic acid, nitric acid, phosphoric acid, p-toluenesulphonicacid, succinic acid, sulfuric acid, sulphuric acid, tartaric acid,trifluoroacetic acid (TFA) and the like. A specific salt istrifluoroacetate.

The terms “pharmaceutically acceptable carrier” and “pharmaceuticallyacceptable auxiliary substance” refer to carriers and auxiliarysubstances such as diluents or excipients that are compatible with theother ingredients of the formulation.

The term “pharmaceutical composition” encompasses a product comprisingspecified ingredients in pre-determined amounts or proportions, as wellas any product that results, directly or indirectly, from combiningspecified ingredients in specified amounts. Preferably it encompasses aproduct comprising one or more active ingredients, and an optionalcarrier comprising inert ingredients, as well as any product thatresults, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients.

The term “half maximal inhibitory concentration” (IC₅₀) denotes theconcentration of a particular compound required for obtaining 50%inhibition of a biological process in vitro. IC₅₀ values can beconverted logarithmically to pIC₅₀ values (−log IC₅₀), in which highervalues indicate exponentially greater potency. The IC₅₀ value is not anabsolute value but depends on experimental conditions e.g.concentrations employed. The IC₅₀ value can be converted to an absoluteinhibition constant (Ki) using the Cheng-Prusoff equation (Biochem.Pharmacol. (1973) 22:3099). The term “inhibition constant” (Ki) denotesthe absolute binding affinity of a particular inhibitor to a receptor.It is measured using competition binding assays and is equal to theconcentration where the particular inhibitor would occupy 50% of thereceptors if no competing ligand (e.g. a radioligand) was present. Kivalues can be converted logarithmically to pKi values (−log Ki), inwhich higher values indicate exponentially greater potency.

“Therapeutically effective amount” means an amount of a compound that,when administered to a subject for treating a disease state, issufficient to effect such treatment for the disease state. The“therapeutically effective amount” will vary depending on the compound,disease state being treated, the severity or the disease treated, theage and relative health of the subject, the route and form ofadministration, the judgment of the attending medical or veterinarypractitioner, and other factors.

The term “as defined herein” and “as described herein” when referring toa variable incorporates by reference the broad definition of thevariable as well as preferred, more preferred and most preferreddefinitions, if any.

The terms “treating”, “contacting” and “reacting” when referring to achemical reaction means adding or mixing two or more reagents underappropriate conditions to produce the indicated and/or the desiredproduct. It should be appreciated that the reaction which produces theindicated and/or the desired product may not necessarily result directlyfrom the combination of two reagents which were initially added, i.e.,there may be one or more intermediates which are produced in the mixturewhich ultimately leads to the formation of the indicated and/or thedesired product.

The invention also provides pharmaceutical compositions, methods ofusing, and methods of preparing the aforementioned compounds.

All separate embodiments may be combined.

Present invention relates to a dual-site BACE1 inhibitor, binding toboth, the enzymatic active site and the catalytic domain of the BACE1enzyme.

A certain embodiment of the invention relates to a dual-site BACE1inhibitor according to claim 1, whereby the exosite inhibitory part (A′)is connected to the active site inhibitory part (B′) of said BACE1inhibitor by a linker (L′), or a pharmaceutically acceptable saltthereof.

A certain embodiment of the invention relates to a dual-site BACE1inhibitor as described herein, wherein L′ is selected from the groupconsisting of

-   -   i. -(Gly)_(x)-, wherein x is 3, 4, 5 or 6(SEQ ID NO: 54),    -   ii. -X-Gly-Gly-, wherein X is selected from the group consisting        of Ala, DAla, Nle, Ser, Pro, D-Pro, Lys and DLys,    -   iii. -Gly-X-Gly-, wherein X is selected from the group        consisting of Ala, DAla, Nle, Ser, Pro, D-Pro, Orn, Lys and        DLys,    -   iv. -Gly-Gly-X-, wherein X is selected from the group consisting        of Ala, DAla, Nle, Ser, Pro, D-Pro, Lys and DLys,    -   v. -Gly-DAla-DLys-,    -   vi. PEG(3), and    -   vii. PEG(4).

A certain embodiment of the invention relates to a compound as describedherein, wherein A′ is selected from the group consisting of

i. (SEQ ID NO: 48) Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-, ii. (SEQ ID NO: 49)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-,  and iii. (SEQ ID NO: 50)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-

A certain embodiment of the invention relates to a compound as describedherein, wherein B′ is selected from the group consisting of i.Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂(SEQ ID NO: 51), ii.Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Y)-NH₂(SEQ ID NO: 52) wherein Y isselected from the group consisting of Palmityl, Palmitoleic, Oleic,Elaidic, Erucic, Vaccenic and Myristoleic, and iii.Glu-Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂(SEQ ID NO: 53), Acertain embodiment of the invention relates to a compound as describedherein, selected from the group consisting of

(SEQ ID NO: 34) Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 36)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palmitoleic)-NH₂, (SEQ ID NO: 38)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Oleic)-NH₂, (SEQ ID NO: 40)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Elaidic)-NH₂, (SEQ ID NO: 42)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Vaccenic)-NH₂, (SEQ ID NO: 44)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Erucic)-NH₂, (SEQ ID NO: 46)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Myristoleic)-NH2, (SEQ ID NO: 35)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 37)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palmitoleic)-NH₂, (SEQ ID NO: 39)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Oleic)-NH₂, (SEQ ID NO: 41)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Elaidic)-NH₂, (SEQ ID NO: 43)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Vaccenic)-NH₂, (SEQ ID NO: 45)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Erucic)-NH₂, (SEQ ID NO: 47)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Myristoleic)-NH₂, (SEQ ID NOS: 33 and 53)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-(PEG)3-Glu-Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂, (SEQ ID NOs: 32 and 53)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-(PEG)4-Glu-Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂, (SEQ ID NO: 2)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Ala-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 11)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DAla-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 27)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DLys-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 18)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DPro-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 3)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Ala-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 21)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DAla-DLys-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 12)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DAla-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 17)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 28)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 19)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DPro-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 4)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Ala-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 13)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-DAla-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 29)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-DLys-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 20)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-DPro-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 1)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 22)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palmityl)-NH₂, (SEQ ID NO: 30)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Gly-Glu-Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂, (SEQ ID NO: 31)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Gly-Gly-Glu-Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)- NH₂, (SEQ ID NO: 26)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Lys-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 7)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Nle-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 16)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Pro-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 10)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Ser-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 23)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Lys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 6)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Nle-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 24)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Orn-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 15)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Pro-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 9)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Ser-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 25)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Lys-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 5)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Nle-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 14)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Pro-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂,  and (SEQ ID NO: 8)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Ser-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂,or a pharmaceutical accpectable salt thereof.

A certain embodiment of the invention relates to a compound as describedherein, wherein the pharmaceutically acceptable salt istrifluoroacetate.

A certain embodiment of the invention relates to a dual-site BACE1inhibitor as described herein for use as therapeutically activesubstance.

A certain embodiment of the invention relates to a dual-site BACE1inhibitor as described herein for the use as inhibitor of BACE1activity.

A certain embodiment of the invention relates to a dual-site BACE1inhibitor as described herein for the use as therapeutically activesubstance for the therapeutic and/or prophylactic treatment of diseasesand disorders characterized by elevated β-amyloid levels and/orβ-amyloid oligomers and/or β-amyloid plaques and further deposits,particularly Alzheimer's disease.

A certain embodiment of the invention relates to a dual-site BACE1inhibitor as described herein for the use as therapeutically activesubstance for the therapeutic and/or prophylactic treatment ofAlzheimer's disease.

A certain embodiment of the invention relates to a pharmaceuticalcomposition comprising a dual-site BACE1 inhibitor as described hereinand a pharmaceutically acceptable carrier and/or a pharmaceuticallyacceptable auxiliary substance.

A certain embodiment of the invention relates to the use of a dual-siteBACE1 inhibitor as described herein for the manufacture of a medicamentfor the use in inhibition of BACE1 activity.

A certain embodiment of the invention relates to the use of a dual-siteBACE1 inhibitor as described herein for the manufacture of a medicamentfor the therapeutic and/or prophylactic treatment of diseases anddisorders characterized by elevated β-amyloid levels and/or β-amyloidoligomers and/or β-amyloid plaques and further deposits, particularlyAlzheimer's disease.

A certain embodiment of the invention relates to the use of a dual-siteBACE1 inhibitor as described herein for the manufacture of a medicamentfor the therapeutic and/or prophylactic treatment of Alzheimer'sdisease.

A certain embodiment of the invention relates to a dual-site BACE1inhibitor as described herein for the use in inhibition of BACE1activity.

A certain embodiment of the invention relates to a dual-site BACE1inhibitor as described herein for the use in the therapeutic and/orprophylactic treatment of diseases and disorders characterized byelevated β-amyloid levels and/or β-amyloid oligomers and/or β-amyloidplaques and further deposits, particularly Alzheimer's disease.

A certain embodiment of the invention relates to a dual-site BACE1inhibitor as described herein for the use in the therapeutic and/orprophylactic treatment of Alzheimer's disease.

A certain embodiment of the invention relates to a method for the use ininhibition of BACE1 activity, particularly for the therapeutic and/orprophylactic treatment of diseases and disorders characterized byelevated β-amyloid levels and/or β-amyloid oligomers and/or β-amyloidplaques and further deposits, Alzheimer's disease, which methodcomprises administering dual-site BACE1 inhibitor as described herein toa human being or animal.

Furthermore, the invention includes all optical isomers, i.e.diastereoisomers, diastereomeric mixtures, racemic mixtures, all theircorresponding enantiomers and/or tautomers as well as their solvates.

The dual-site BACE1 inhibitors may be prepared as described herein. Thestarting material is commercially available or may be prepared inaccordance with known methods.

The corresponding pharmaceutically acceptable salts with acids can beobtained by standard methods known to the person skilled in the art,e.g. by dissolving the dual-site BACE1 inhibitor in a suitable solventsuch as e.g. dioxan or THF and adding an appropriate amount of thecorresponding acid. The products can usually be isolated by filtrationor by chromatography. The conversion of a dual-site BACE1 inhibitor intoa pharmaceutically acceptable salt with a base can be carried out bytreatment of such a compound with such a base. One possible method toform such a salt is e.g. by addition of 1/n equivalents of a basic saltsuch as e.g. M(OH)_(n), wherein M=metal or ammonium cation and n=numberof hydroxide anions, to a solution of the compound in a suitable solvent(e.g. ethanol, ethanol-water mixture, tetrahydrofuran-water mixture) andto remove the solvent by evaporation or lyophilisation.

Insofar as their preparation is not described in the examples, thedual-site BACE1 inhibitors as well as all intermediate products can beprepared according to analogous methods or according to the methods setforth herewithin. Starting materials are commercially available, knownin the art or can be prepared by methods known in the art or in analogythereto.

It will be appreciated that the dual-site BACE1 inhibitors in thisinvention may be derivatised at functional groups to provide derivativeswhich are capable of conversion back to the parent compound in vivo.

Pharmacological Tests

The dual-site BACE1 inhibitor and their pharmaceutically acceptablesalts possess valuable pharmacological properties. It has been foundthat the compounds of the present invention are associated withinhibition of BACE1 activity. The compounds were investigated inaccordance with the test given hereinafter.

Cellular Aβ-lowering Assay:

Human HEK293 cells which are stably transfected with a vector expressinga cDNA of the human APP wt gene (APP695) were used to assess the potencyof the compounds in a cellular assay. The cells were seeded in 96-wellmicrotiter plates in cell culture medium (Iscove, plus 10% (v/v) fetalbovine serum, glutamine, penicillin/streptomycin) to about 80%confluence and the compounds were added at a 10× concentration in 1/10volume of medium without FCS containing 8% DMSO (final concentration ofDMSO was kept at 0.8% v/v). After 18-20 hrs incubation at 37° C. and 5%CO₂ in a humidified incubator the culture supernatant was harvested forthe determination of Aβ40 concentrations. 96well ELISA plates (e.g.,Nunc MaxiSorb) were coated with monoclonal antibody which specificallyrecognize the C-terminal end of Aβ40 (Brockhaus et al., NeuroReport 9,1481-1486; 1998). After blocking of non-specific binding sites with e.g.1% BSA and washing, the culture supernatants were added in suitabledilutions together with a horseradish peroxidase-coupled Aβ detectionantibody (e.g., antibody 4G8, Senetek, Maryland Heights, Mo.) andincubated for 5 to 7 hrs. Subsequently the wells of the microtiter platewere washed extensively with Tris-buffered saline containing 0.05% Tween20 and the assay was developed with tetramethylbenzidine/H₂O₂ in citricacid buffer. After stopping the reaction with one volume 1 N H₂SO₄ thereaction was measured in an ELISA reader at 450 nm wavelength. Theconcentrations of Aβ in the culture supernatants were calculated from astandard curve obtained with known amounts of pure Aβ peptide.

TABLE 2 IC₅₀ values of selected examples SEQ ID IC 50 NO: Ex. NameSystematic Name MW (μM)  1  1 YPYFIPL-GGG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2508.9 0.12 Sta-VAEFK(Palm)-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA  2  2 YPYFIPL-AGG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Ala- 2523.9 0.053 Sta-VAEFK(Palm)-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA  3  3 YPYFIPL-GAG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2522.9 0.095 Sta-VAEFK(Palm)-Ala-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA  4  4 YPYFIPL-GGA-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2522.9 0.084 Sta-VAEFK(Palm)-Gly-Ala-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA  5  5 YPYFIPL-Nle-GG-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Nle- 2565.0 0.081 EVN-Sta-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- VAEFK(Palm)-NH₂ xGlu-Phe-Lys(Palmityl)-NH₂ x TFA TFA  6  6 YPYFIPL-G-N1e-G-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2565.0 0.12 EVN-Sta-Nle-Gly-Glu-Val-Asn-Sta-Val-Ala- VAEFK(Palm)-NH₂ xGlu-Phe-Lys(Palmityl)-NH₂ x TFA TFA  7  7 YPYFIPL-GG-Nle-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2565.0 0.18 EVN-Sta-Gly-Nle-Glu-Val-Asn-Sta-Val-Ala- VAEFK(Palm)-NH₂ xGlu-Phe-Lys(Palmityl)-NH₂ x TFA TFA  8  8 YPYFIPL-SGG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Ser- 2538.9 0.036 Sta-VAEFK(Palm)-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA  9  9 YPYFIPL-GSG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2538.9 0.041 Sta-VAEFK(Palm)-Ser-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA 10 10 YPYFIPL-GGS-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2538.9 0.053 Sta-VAEFK(Palm)-Gly-Ser-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA 11 11 YPYFIPL-aGG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DAla- 2522.9 0.013 Sta-VAEFK(Palm)-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA 12 12 YPYFIPL-GaG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2522.9 0.0086 Sta-VAEFK(Palm)-DAla-Gly-Glu-Val-Asn-Sta-Val- NH₂ x TFA Ala-Glu-Phe-Lys(Palmityl)-NH₂ xTFA 13 13 YPYFIPL-GGa-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2522.9 0.025Sta-VAEFK(Palm)- Gly-DAla-Glu-Val-Asn-Sta-Val- NH₂ x TFAAla-Glu-Phe-Lys(Palmityl)-NH2 x TFA 14 14 YPYFIPL-PGG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Pro- 2549.0 0.021 Sta-VAEFK(Palm)-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA 15 15 YPYFIPL-GPG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2549.0 0.14 Sta-VAEFK(Palm)-Pro-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA 16 16 YPYFIPL-GGP-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2549.0 0.049 Sta-VAEFK(Palm)-Gly-Pro-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA 17 17 YPYFIPL-GkG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2580.0 0.064 Sta-VAEFK(Palm)-DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x TFA Ala-Glu-Phe-Lys(Palmityl)-NH₂ xTFA 18 18 YPYFIPL-pGG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DPro- 2549.00.050 Sta-VAEFK(Palm)- Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA 19 19 YPYFIPL-GpG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2549.0 0.048 Sta-VAEFK(Palm)-DPro-Gly-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA 20 20 YPYFIPL-GGp-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2549.0 0.11 Sta-VAEFK(Palm)-Gly-DPro-Glu-Val-Asn-Sta-Val-Ala- NH₂ x TFAGlu-Phe-Lys(Palmityl)-NH₂ x TFA 21 21 YPYFIPL-Gak-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2594.1 0.098 Sta-VAEFK(Palm)-DAla-DLys-Glu-Val-Asn-Sta-Val- NH₂ x TFA Ala-Glu-Phe-Lys(Palmityl)-NH₂ xTFA 22 22 YPYFIPL-GGG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2458.9 0.060Sta-VAEpK(Palm)-NH₂ Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-  x TFAGlu-DPro-Lys(Palmityl)-NH₂ x TFA 23 23 YPYFIPL-GKG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2644.0 0.078 Sta-VAE-DPro-Lys-Gly-Glu-Val-Asn-Sta-Val-Ala- K(Palm)-NH2 x 2TFAGlu-DPro-Lys(Palm)-NH₂ x 2TFA 24 24 YPYFIPL-GOG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2630.0 0.067 Sta-VAE-DPro-Orn-Gly-Glu-Val-Asn-Sta-Val-Ala- K(Palm)-NH2 x 2TFAGlu-DPro-Lys(Palm)-NH₂ x 2TFA 25 25 YPYFIPL-KGG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Lys- 2644.0 0.0039 Sta-VAE-DPro-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- K(Palm)-NH₂ x 2TFAGlu-DPro-Lys(Palm)-NH₂ x 2TFA 26 26 YPYFIPL-GGK-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2644.0 0.0033 Sta-VAE-DPro-Gly-Lys-Glu-Val-Asn-Sta-Val-Ala- K(Palm)-NH₂ x 2TFAGlu-DPro-Lys(Palm)-NH₂ x 2TFA 27 27 YPYFIPL-kGG-EVN-Tyr-Pro-Tyr-Phe-fle-Pro-Leu-DLys- 2644.0 0.0060 Sta-VAE-DPro-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala- K(Palm)-NH₂ x 2TFAGlu-DPro-Lys(Palm)-NH₂ x 2TFA 28 28 YPYFIPL-GkG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2644.0 0.0029 Sta-VAE-DPro-DLys-Gly-Glu-Val-Asn-Sta-Val- K(Palm)-NH₂ x 2TFAAla-Glu-DPro-Lys(Palm)-NH₂ x 2TFA 29 29 YPYFIPL-GGk-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2644.0 0.0057 Sta-VAE-DPro-Gly-DLys-Glu-Val-Asn-Sta-Val- K(Palm)-NH₂ x 2TFAAla-Glu-DPro-Lys(Palm).NH₂ x 2TFA 30 30 YPYFIPL-GGGG-Tyr-Pro-Tyr-Phe-fle-Pro-Leu-Gly- 2584.0 0.33 EVN-MetSta-VAE-Gly-Gly-Gly-Glu-Val-Asn-MetSta- DPhe-K(Palm)-NH₂ xVal-Ala-Glu-DPhe-Lys(Palm)-NH₂ x TFA TFA 31 31 YPYFIPL-GGGGG-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly- 2641.1 0.034 EVN-MetSta-VAE-Gly-Gly-Gly-Gly-Glu-Val-Asn- DPhe-K(Palm)-NH₂ x MetSta-Val-Ala-Glu-DPhe-TFA Lys(Palm)-NH₂ x TFA 32 & 32 YPYFIPL-PEG(4)-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu- 2603.1 0.018 53 EVN-MetSta-VAE-(PEG)4-Glu-Val-Asn-MetSta-Val- DPhe-K(Palm)-NH₂ xAla-Glu-DPhe-Lys(Palm)-NH₂ x TFA TFA 33 & 33 YPYFIPL-PEG(3)-Tyr-Pro-Tyr-Phe-Ile-Pro-Leu- 2559.0 0.041 53 EVN-MetSta-VAE-(PEG)3-Glu-Val-Asn-MetSta-Val- DPhe-K(Palm)-NH₂ xAla-Glu-DPhe-Lys(Palm)-NH₂ x TFA TFA 34 34 YPKFIPL-GkG-EVN-Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2723.0 0.00137 Sta-VAEp-K(Palm)-DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFA Ala-Glu-DPro-Lys(Palm)-NH₂ x3TFA 35 35 YPYFIPk-GkG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2773.00.000199 Sta-VAEp-K(Palm)- DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFAAla-Glu-DPro-Lys(Palm)-NH₂ x 3TFA 36 36 YPKFIPL-GkG-EVN-Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2721.0 0.0199 Sta-VAEp-KDLys-Gly-Glu-Val-Asn-Sta-Val- (Palmitoleic)-NH₂ xAla-Glu-DPro-Lys(Palmitoleic)-NH₂ 3TFA x 3TFA 37 37 YPYFIPk-GkG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2771.0 0.00428 Sta-VAEp-DLys-Gly-Glu-Val-Asn-Sta-Val- K(Palmitoleic)-NH₂ xAla-Glu-DPro-Lys(Palmitoleic)-NH₂ 3TFA x 3TFA 38 38 YPKFIPL-GkG-EVN-Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2749.1 0.00602 Sta-VAEp-K(Oleic)-DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFA Ala-Glu-DPro-Lys(Oleic)-NH₂ x3TFA 39 39 YPYFIPk-GkG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2799.10.00142 Sta-VAEp-K(Oleic)- DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFAAla-Glu-DPro-Lys(Oleic)-NH₂ x 3TFA 40 40 YPKFIPL-GkG-EVN-Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2749.1 0.00302 Sta-VAEp-K(Elaidic)-DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFA Ala-Glu-DPro-Lys(Elaidic)-NH₂ x3TFA 41 41 YPYFIPk-GkG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2799.10.000906 Sta-VAEp-K(Elaidic)- DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFAAla-Glu-DPro-Lys(Elaidic)-NH₂ x 3TFA 42 42 YPKFIPL-GkG-EVN-Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2749.1 0.00268 Sta-VAEp-KDLys-Gly-Glu-Val-Asn-Sta-Val- (Vaccenic)-NH₂ xAla-Glu-DPro-Lys(Vaccenic)-NH₂ x 3TFA 3TFA 43 43 YPYFIPk-GkG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2799.1 0.00111 Sta-VAEp-DLys-Gly-Glu-Val-Asn-Sta-Val- K(Vaccenic)-NH₂ xAla-Glu-DPro-Lys(Vaccenic)-NH₂ x 3TFA 3TFA 44 44 YPKFIPL-GkG-EVN-Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2805.2 0.00115 Sta-VAEp-K(Erucic)-DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFA Ala-Glu-DPro-Lys(Erucic)-NH₂ x3TFA 45 45 YPYFIPk-GkG-EVN- Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2855.20.000147 Sta-VAEp-K(Erucic)- DLys-Gly-Glu-Val-Asn-Sta-Val- NH₂ x 3TFAAla-Glu-DPro-Lys(Erucic)-NH₂ x 3TFA 46 46 YPKFIPL-GkG-EVN-Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly- 2693.0 0.208 Sta-VAEp-KDLys-Gly-Glu-Val-Asn-Sta-Val- (Myristoleic)-NH₂ xAla-Glu-DPro-Lys(Myristoleic)-NH₂ 3TFA  x 3TFA 47 47 YPYFIPk-GkG-EVN-Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly- 2743.0 0.0196 Sta-VAEp-KDLys-Gly-Glu-Val-Asn-Sta-Val- (Myristoleic)-NH₂ xAla-Glu-DPro-Lys(Myristoleic)-NH₂ 3TFA  x 3TFAPharmaceutical Compositions

The dual-site BACE1 inhibitors and the pharmaceutically acceptable saltscan be used as therapeutically active substances, e.g. in the form ofpharmaceutical preparations. Examples for pharmaceutical preparationsare an enteral formulation, e.g. in the form of tablets, coated tablets,dragées, hard and soft gelatine capsules, solutions, emulsions orsuspensions or the like. The administration can, however, also beeffected rectally, e.g. in the form of suppositories, parenterally, e.g.in the form of injection solutions, or as intranasal delivery, e.g. asnasal spray.

The dual-site BACE1 inhibitors and the pharmaceutically acceptable saltsthereof can be processed with pharmaceutically inert, inorganic ororganic carriers for the production of pharmaceutical preparations.Lactose, corn starch or derivatives thereof, talc, stearic acids or itssalts and the like can be used, for example, as such carriers fortablets, coated tablets, dragées and hard gelatine capsules. Suitablecarriers for soft gelatine capsules are, for example, vegetable oils,waxes, fats, semi-solid and liquid polyols and the like. Depending onthe nature of the active substance no carriers are however usuallyrequired in the case of soft gelatine capsules. Suitable carriers forthe production of solutions and syrups are, for example, water, polyols,glycerol, vegetable oil and the like. Suitable carriers forsuppositories are, for example, natural or hardened oils, waxes, fats,semi-liquid or liquid polyols and the like. The dual-site BACE1inhibitors and the pharmaceutically acceptable salts thereof can also beencapsulated in suitable polymers or formulated using nanotechnology.

The pharmaceutical preparations can, moreover, contain pharmaceuticallyacceptable auxiliary substances such as preservatives, solubilizers,stabilizers, wetting agents, emulsifiers, sweeteners, colorants,flavorants, salts for varying the osmotic pressure, buffers, maskingagents or antioxidants. They can also contain still othertherapeutically valuable substances.

Medicaments containing a dual-site BACE1 inhibitor or a pharmaceuticallyacceptable salt thereof and a therapeutically inert carrier are also anobject of the present invention, as is a process for their production,which comprises bringing one or more dual-site BACE1 inhibitors and/orpharmaceutically acceptable salts thereof and, if desired, one or moreother therapeutically valuable substances into a galenicaladministration form together with one or more therapeutically inertcarriers.

The dosage can vary within wide limits and will, of course, have to beadjusted to the individual requirements in each particular case. In thecase of oral administration the dosage for adults can vary from about0.01 mg to about 1000 mg per day of a dual-site BACE1 inhibitors or ofthe corresponding amount of a pharmaceutically acceptable salt thereof.The daily dosage may be administered as single dose or in divided dosesand, in addition, the upper limit can also be exceeded when this isfound to be indicated.

The following examples illustrate the present invention without limitingit, but serve merely as representative thereof. The pharmaceuticalpreparations conveniently contain about 1-500 mg, preferably 1-100 mg,of a dual-site BACE1 inhibitor. Examples of compositions according tothe invention are:

EXAMPLE A

Tablets of the following composition are manufactured in the usualmanner:

TABLE 3 possible tablet composition mg/tablet ingredient 5 25 100 500Dual-site BACE1 inhibitor 5 25 100 500 Lactose Anhydrous DTG 125 105 30150 Sta-Rx 1500 6 6 6 60 Microcrystalline Cellulose 30 30 30 450Magnesium Stearate 1 1 1 1 Total 167 167 167 831Manufacturing Procedure

-   1. Mix ingredients 1, 2, 3 and 4 and granulate with purified water.-   2. Dry the granules at 50° C.-   3. Pass the granules through suitable milling equipment.-   4. Add ingredient 5 and mix for three minutes; compress on a    suitable press.

Example B-1

Capsules of the following composition are manufactured:

TABLE 4 possible capsule ingredient composition mg/capsule ingredient 525 100 500 Dual-site BACE1 inhibitor 5 25 100 500 Hydrous Lactose 159123 148 — Corn Starch 25 35 40 70 Talk 10 15 10 25 Magnesium Stearate 12 2 5 Total 200 200 300 600Manufacturing Procedure

-   1. Mix ingredients 1, 2 and 3 in a suitable mixer for 30 minutes.-   2. Add ingredients 4 and 5 and mix for 3 minutes.-   3. Fill into a suitable capsule.

The dual-site BACE1 inhibitor, lactose and corn starch are firstly mixedin a mixer and then in a comminuting machine. The mixture is returned tothe mixer; the talc is added thereto and mixed thoroughly. The mixtureis filled by machine into suitable capsules, e.g. hard gelatinecapsules.

Example B-2

Soft Gelatine Capsules of the following composition are manufactured:

TABLE 5 possible soft gelatin capsule ingredient composition ingredientmg/capsule Dual-site BACE1 inhibitor 5 Yellow wax 8 Hydrogenated Soyabean oil 8 Partially hydrogenated plant oils 34 Soya bean oil 110 Total165

TABLE 6 possible soft gelatin capsule composition ingredient mg/capsuleGelatin 75 Glycerol 85% 32 Karion 83 8 (dry matter) Titan dioxide 0.4Iron oxide yellow 1.1 Total 116.5Manufacturing Procedure

The dual-site BACE1 inhibitor is dissolved in a warm melting of theother ingredients and the mixture is filled into soft gelatin capsulesof appropriate size. The filled soft gelatin capsules are treatedaccording to the usual procedures.

Example C

Suppositories of the following composition are manufactured:

TABLE 7 possible suppository composition ingredient mg/supp. Dual-siteBACE1 inhibitor 15 Suppository mass 1285 Total 1300Manufacturing Procedure

The suppository mass is melted in a glass or steel vessel, mixedthoroughly and cooled to 45° C. Thereupon, the finely powdered dual-siteBACE1 inhibitor is added thereto and stirred until it has dispersedcompletely. The mixture is poured into suppository moulds of suitablesize, left to cool; the suppositories are then removed from the mouldsand packed individually in wax paper or metal foil.

Example D

Injection solutions of the following composition are manufactured:

TABLE 8 possible injection solution composition ingredient mg/injectionsolution. Dual-site BACE1 inhibitor  3 Polyethylene Glycol 400 150acetic acid q.s. ad pH 5.0 water for injection solutions ad 1.0 mlManufacturing Procedure

The dual-site BACE1 inhibitor is dissolved in a mixture of PolyethyleneGlycol 400 and water for injection (part). The pH is adjusted to 5.0 byacetic acid. The volume is adjusted to 1.0 ml by addition of theresidual amount of water. The solution is filtered, filled into vialsusing an appropriate overage and sterilized.

Example E

Sachets of the following composition are manufactured:

TABLE 9 possible sachet composition ingredient mg/sachet Dual-site BACE1inhibitor 50 Lactose, fine powder 1015 Microcrystalline cellulose(AVICEL PH 102) 1400 Sodium carboxymethyl cellulose 14Polyvinylpyrrolidon K 30 10 Magnesium stearate 10 Flavoring additives 1Total 2500Manufacturing Procedure

The dual-site BACE1 inhibitor is mixed with lactose, microcrystallinecellulose and sodium carboxymethyl cellulose and granulated with amixture of polyvinylpyrrolidone in water. The granulate is mixed withmagnesium stearate and the flavoring additives and filled into sachets.

Experimental Part

The following examples are provided for illustration of the invention.They should not be considered as limiting the scope of the invention,but merely as being representative thereof.

General Procedures for the CEM Liberty Microwave Peptide Synthesizer:

0.1 mMol scale:

Deprotection of Fmoc:

The washed and preswelled resin (435 mg, 0.1 mMol, TentaGel S RAM (Load:0.23 mMol/g), (Rapp Polymere, Cat: S30023) was treated with a solutionof piperidine 20% in dimethylformamide (DMF) (7.0 mL) under microwavecondition at 50° C. for 3 minutes for initial deprotection. The resinwas washed with DMF and treated with a solution of piperidine 20% in DMF(7.0 mL) under microwave condition at 75° C. for 5 minutes fordeprotection.

Coupling of amino acids:

To the washed and preswelled resin was added a solution of amino acid,0.2M in DMF (2.5 mL, 5.0 eq.) followed by a solution of COMU® 0.5M inDMF (1.0 mL, 5.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat:RL-1175.1000) followed by a solution of diisopropylethylamine (DIPEA) 2Min N-Methyl-2-pyrrolidone (NMP) (0.5 mL, 10.0 eq.). This reactionmixture was treated under microwave condition at 75° C. for 5 minutesfor coupling.

0.25 mMol scale:

Deprotection of Fmoc:

The washed and preswelled resin (1.09 g, 0.25 mMol, TentaGel S RAM(Load: 0.23 mMol/g), (Rapp Polymere, Cat: S30023) was treated with asolution of piperidine 20% in DMF (10.0 mL) under microwave condition at50° C. for 3 minutes for initial deprotection. The resin was washed withDMF and treated with a solution of piperidine 20% in DMF (10.0 mL) undermicrowave condition at 75° C. for 5 minutes for deprotection.

Coupling of amino acids:

To the washed and preswelled resin was added a solution of amino acid,0.2M in DMF (5.0 mL, 4.0 eq.) followed by a solution of COMU® 0.5M inDMF (2.0 mL, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat:RL-1175.1000) followed by a solution of DIPEA 2M in NMP (1.0 mL, 8.0eq.). This reaction mixture was treated under microwave condition at 75°C. for 5 minutes for coupling.

General Procedure for Coupling of “n-C8”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with asolution of caprylic acid (79.2 μl, 5.0 eq.), (CAS: 124-07-2, Fluka) andCOMU® (214 mg, 5.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat:RL-1175.1000) and DIPEA (204 μL, 12.0 eq.) in 5.0 mL DMF for 1 hour atroom temperature on the shaker.

General Procedure for Coupling of “n-C12”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with asolution of dodecanoic acid (100 mg, 5.0 eq.), (CAS: 143-07-7, Aldrich)and COMU® (214 mg, 5.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat:RL-1175.1000) and DIPEA (204 μL, 12.0 eq.) in 5.0 mL DMF for 1 hour atroom temperature on the shaker.

General Procedure for Coupling of “n-C14”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with asolution of myristic acid (114 mg, 5.0 eq.), (CAS: 544-63-8, Fluka) andCOMU® (214 mg, 5.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat:RL-1175.1000) and DIPEA (204 μL, 12.0 eq.) in 5.0 mL DMF for 1 hour atroom temperature on the shaker.

General Procedure for Coupling of “Palmitoleic”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with asolution of palmitoleic acid (102 mg, 4.0 eq.), (CAS: 373-49-9) andCOMU® (176 mg, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat:RL-1175.1000) and DIPEA (136 μL, 8.0 eq.) in 5.0 mL DMF for 1 hour atroom temperature on the shaker.

General Procedure for Coupling of “Oleic”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with asolution of oleic acid (113 mg, 4.0 eq.), (CAS: 112-80-1) and COMU® (176mg, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000) andDIPEA (136 μL, 8.0 eq.) in 5.0 mL DMF for 1 hour at room temperature onthe shaker.

General Procedure for Coupling of “Elaidic”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with asolution of elaidic acid (113 mg, 4.0 eq.), (CAS: 112-79-8) and COMU®(176 mg, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000)and DIPEA (136 μL, 8.0 eq.) in 5.0 mL DMF for 1 hour at room temperatureon the shaker.

General Procedure for Coupling of “Vaccenic”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with asolution of vaccenic acid (113 mg, 4.0 eq.), (CAS: 693-72-1) and COMU®(176 mg, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000)and DIPEA (136 μL, 8.0 eq.) in 5.0 mL DMF for 1 hour at room temperatureon the shaker.

General Procedure for Coupling of “Erucic”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with asolution of erucic acid (113 mg, 4.0 eq.), (CAS: 112-86-7) and COMU®(176 mg, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat: RL-1175.1000)and DIPEA (136 μL, 8.0 eq.) in 5.0 mL DMF for 1 hour at room temperatureon the shaker.

General Procedure for Coupling of “Myristoleic”:

0.1 mMol scale:

The deprotected, with DMF washed and preswelled resin was treated with asolution of myristoleic acid (90.5 mg, 4.0 eq.), (CAS: 544-64-9) andCOMU® (176 mg, 4.0 eq.), (CAS: 1075198-30-9, Iris Biotech, Cat:RL-1175.1000) and DIPEA (136 μL, 8.0 eq.) in 5.0 mL DMF for 1 hour atroom temperature on the shaker.

General Procedure for Final Cleavage:

0.1 mMol scale:

The resin was washed with dichloromethane (CH₂Cl₂) and then treated witha solution of trifluoroacetic acid (TFA): TIS:water 95:2.5:2.5 (5 mL)for 30 minutes at room temperature on the shaker. The resin wasfiltered. The crude peptide was precipitated with diethylether (Et₂O)(35 mL). The suspension was centrifuged and the solvent was decanted.The solid was dissolved in acetonitrile and water and freeze-dried toget the crude peptide.

General Procedure for Purification:

The crude product was dissolved in acetonitrile and water (containing0.1% TFA) and then purified by preparative HPLC. Column YMC-Actus ProC8, 5 μm, 75×30 mm with a gradient of water (containing 0.1%TFA):acetonitrile 70:30 to 2:98 and with a flow of 30 mL/min.

All Examples as described in table 1 can be prepared analogous to thegeneral procedures described herein.

The invention claimed is:
 1. A dual-site BACE1 inhibitor, selected fromthe group consisting of: (SEQ ID NO: 34)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 36)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palmitoleic)-NH₂, (SEQ ID NO: 38)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Oleic)-NH₂, (SEQ ID NO: 40)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Elaidic)-NH₂, (SEQ ID NO: 42)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Vaccenic)-NH₂, (SEQ ID NO: 44)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Erucic)-NH₂, (SEQ ID NO: 46)Tyr-Pro-Lys-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Myristoleic)-NH2, (SEQ ID NO: 35)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 37)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palmitoleic)-NH₂, (SEQ ID NO: 39)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Oleic)-NH₂, (SEQ ID NO: 41)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Elaidic)-NH₂, (SEQ ID NO: 43)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Vaccenic)-NH₂, (SEQ ID NO: 45)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Erucic)-NH₂, (SEQ ID NO: 47)Tyr-Pro-Tyr-Phe-Ile-Pro-DLys-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Myristoleic)-NH₂, (SEQ ID NOS: 33 and 53)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-(PEG)3-Glu-Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂, (SEQ ID NOs: 32 and 53)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-(PEG)4-Glu-Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂, (SEQ ID NO: 2)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Ala-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 11)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DAla-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 27)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DLys-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 18)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-DPro-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 3)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Ala-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 21)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DAla-DLys-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 12)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DAla-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 17)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 28)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DLys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 19)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-DPro-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 4)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Ala-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 13)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-DAla-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 29)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-DLys-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 20)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-DPro-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 1)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 22)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palmityl)-NH₂, (SEQ ID NO: 30)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Gly-Glu-Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)-NH₂, (SEQ ID NO: 31)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Gly-Gly-Gly-Glu-Val-Asn-MetSta-Val-Ala-Glu-DPhe-Lys(Palm)- NH₂, (SEQ ID NO: 26)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Lys-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 7)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Nle-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 16)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Pro-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 10)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Gly-Ser-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 23)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Lys-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 6)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Nle-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 24)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Orn-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 15)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Pro-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 9)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Gly-Ser-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 25)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Lys-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-DPro-Lys(Palm)-NH₂, (SEQ ID NO: 5)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Nle-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂, (SEQ ID NO: 14)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Pro-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂,  and (SEQ ID NO: 8)Tyr-Pro-Tyr-Phe-Ile-Pro-Leu-Ser-Gly-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Lys(Palmityl)-NH₂,

or a pharmaceutically acceptable salt thereof.
 2. The dual-site BACE1inhibitor according to claim 1, wherein the pharmaceutically acceptablesalt is trifluoroacetate.
 3. A pharmaceutical composition, comprising adual-site BACE1inhibitor according to claim 1 and a pharmaceuticallyacceptable carrier and/or a pharmaceutically acceptable auxiliarysubstance.
 4. A method of inhibiting BACE1 activity, comprising the stepof administering a dual-site BACE1 inhibitor according to claim 1 to ahuman being or animal in need thereof.
 5. A method of treating a diseaseor disorder characterized by elevated β-amyloid levels and/or β-amyloidoligomers and/or β-amyloid plaques and further deposits, comprising thestep of administering a dual-site BACE1 inhibitor according to claim 1to a human being or animal in need thereof.
 6. The method according toclaim 5, wherein said disease or disorder is Alzheimer's disease.